The present invention relates to a method and apparatus for magnetically separating particles of a selected type (hereinafter called xe2x80x9ctarget particlesxe2x80x9d) from a sample in order to produce a concentration of the target particles in the sample, and or a depletion of the sample with respect to the target particles. The invention is particularly useful for magnetically separating biological cells of a selected type, e.g., a selected type of lymphocyte cell in a blood sample, and is therefore described below especially with respect to such applications.
A large number of applications involving the magnetic separation of biological cells are described in the literature, for example in U.S. Pat. No. 4,710,472 and the many publications cited therein, which are hereby incorporated by reference. Many such applications require not only the separation of one or more specific types of cells (hereinafter called xe2x80x9ctarget cellsxe2x80x9d), but also the maintenance of the quality of the cell membranes in the target cells, and/or in the untargetted cells. Thus, in a positive selection process, the target cells are separated from a sample for examination or use for research, diagnostic or clinical purposes; whereas in a depletion process, the sample is depleted of the target cells for examination or use of the untargetted cells. The separation of target cells from the untargetted cells, and the maintenance of the membranes of both the target cells and untargetted cells, are particularly important in research presently being conducted with lymphocyte populations and their role in the early detection of cancer.
One technique in present use for the separation of biological cells utilizes the MiniMACS Separation Columns (Miltenyi Biotec GmbH). This technique uses paramagnetic microbeads which are extremely small, about 50 nm in diameter, i.e., about one milion times smaller in volume than that of eukatyotic cells, compared to the size of a virus. Such magnetic microbeads are produced with selective affinities for certain cells, i.e., the target cells, such that they magnetically label or stain the target cells. The sample is introduced into a magnetic separation column including a liquid-pervious magnetic body, e.g., steel wool or mesh, and a magnetic field is applied across the column such that the magnetically stained cells are retained in the liquid-pervious magnetic body of the column, while the unstained cells pass through the column. In this known process, however, it was found that the membranes of the cells are excessively damaged by the liquid-pervious magnetic body, which reduces the effectiveness of the technique for research or clinical purposes.
An object of the present invention is to provide a method of magnetically separating target particles of a selected type from a sample in a manner which causes less damage to the membrane than the above described known technique. Another object of the present invention is to provide apparatus for magnetically separating target particles in accordance with the novel method.
According to one aspect of the present invention, there is provided a method of magnetically separating target particles of a selected type from a sample in order to produce a concentration of the target particles in the sample, or a depletion of the sample with respect to the target particles, comprising: producing a sample mixture of the sample with magnetic particles having a selective affinity to magnetically stain the target particles; producing a flow of a buffer liquid through a tube which includes an inlet connectable to a source of buffer liquid, and an outlet for the buffer liquid; after a flow of the buffer liquid has been produced through the tube, introducing the sample mixture into the buffer liquid flowing through the tube such that the buffer liquid forms a continuous liquid carrier for the sample mixture as both are fed through the tube; and applying a magnetic field across the tube at a magnetizing station therein to cause the magnetically-stained target particles to be separated and retained in the buffer liquid within the tube at the magnetizing station.
Such a method is particularly useful in a depletion process, wherein a sample depleted of the target particles is to be produced for diagnostic examination, research, or clinical purposes.
According to further features in the described preferred embodiments, the magnetically-stained target particles in the sample mixture, which are separated and retained in the buffer liquid within the tube at the magnetizing station, are subsequently removed from the tube by terminating the introduction of the sample mixture into the buffer liquid and the application of the magnetic field across the tube, while the buffer liquid is fed through the tube to flush out the magnetically-stained target particles with the buffer liquid. Such a method is particularly useful in a positive selection process, wherein the target particles are to be separated and used for diagnostic examination, research or clinical purposes.
According to another aspect of the present invention, there is provided apparatus for magnetically separating target particles of a selected type from a sample in order to produce a concentration of the target particles in the sample, or a depletion of the sample with respect to the target particles, comprising: a buffer liquid supply; a tube for feeding a buffer liquid from the buffer liquid supply at an inlet end of the tube to an outlet end of the tube; a sample container for containing a mixture of the sample with magnetic particles having a selective affinity to magnetically stain the target particles; a feed tube connecting the container to the inlet end of the tube to enable feeding the mixture through the tube after a flow of buffer liquid has been produced therein, such that the buffer liquid forms a continuous liquid carrier for the magnetically-stained target particles of the mixture fed through the tube; magnetic field producing means for producing a magnetic field across the tube at a magnetizing station therein to cause the magnetically-stained target particles to be separated and retained in the buffer liquid within the tube at the magnetizing station; and a container located at the outlet end of the tube for receiving the buffer liquid and the sample depleted of the target particles.
Where the apparatus is to be used in a positive selection process, the apparatus further comprises a second container which can be located at the outlet end of the tube in place of the first-mentioned container; in addition, the application of the magnetic field, and the inputting of the mixture into the buffer liquid, are both terminated to cause the buffer liquid fed through the tube to flush out the magnetically-stained target particles into the second container.
Such a method and apparatus have been found to enable the separation of selected types of particles, (target particles), particularly biological cells (target cells), without causing undue damage to either the target particles or the untargetted particles. Thus, the buffer liquid, which forms a continuous liquid carrier for both the target particles and the untargetted particles, produces a constant liquid volume which physically supports both types of particles (or cells) during both phases of the process, thereby minimizing damage to both types of particles during both phases.
While the method and apparatus of the present invention are particularly useful for separating selected types of biological cells, such method and apparatus may also be used for separating other types of particles, e.g., selected proteins. Also, while the described method and apparatus preferable use the commercially-available magnetic microbeads, it will be appreciated that other magnetic particles having a selective affinity for the target particles may be used to magnetically stain or label the target particles.
Further features and advantages of the invention will be apparent from the description below.